toxins

Review
Colubrid Venom Composition:
An -Omics Perspective
Inácio L. M. Junqueira-de-Azevedo 1, *, Pollyanna F. Campos 1 , Ana T. C. Ching 2 and
Stephen P. Mackessy 3
1 Laboratório Especial de Toxinologia Aplicada, Center of Toxins, Immune-Response and Cell
Signaling (CeTICS), Instituto Butantan, São Paulo 05503-900, Brazil; pollyanna.fc@gmail.com
2 Laboratório de Imunoquímica, Instituto Butantan, São Paulo 05503-900, Brazil; anatung@gmail.com
3 School of Biological Sciences, University of Northern Colorado, Greeley, CO 80639-0017, USA;
stephen.mackessy@unco.edu
* Correspondence: inacio.azevedo@butantan.gov.br; Tel.: +55-11-2627-9731

Academic Editor: Bryan Fry

Received: 7 June 2016; Accepted: 8 July 2016; Published: 23 July 2016

Abstract: Snake venoms have been subjected to increasingly sensitive analyses for well over 100 years,
but most research has been restricted to front-fanged snakes, which actually represent a relatively
small proportion of extant species of advanced snakes. Because rear-fanged snakes are a diverse and
distinct radiation of the advanced snakes, understanding venom composition among “colubrids”
is critical to understanding the evolution of venom among snakes. Here we review the state of
knowledge concerning rear-fanged snake venom composition, emphasizing those toxins for which
protein or transcript sequences are available. We have also added new transcriptome-based data on
venoms of three species of rear-fanged snakes. Based on this compilation, it is apparent that several
components, including cysteine-rich secretory proteins (CRiSPs), C-type lectins (CTLs), CTLs-like
proteins and snake venom metalloproteinases (SVMPs), are broadly distributed among “colubrid”
venoms, while others, notably three-finger toxins (3FTxs), appear nearly restricted to the Colubridae
(sensu stricto). Some putative new toxins, such as snake venom matrix metalloproteinases, are in
fact present in several colubrid venoms, while others are only transcribed, at lower levels. This work
provides insights into the evolution of these toxin classes, but because only a small number of species
have been explored, generalizations are still rather limited. It is likely that new venom protein
families await discovery, particularly among those species with highly specialized diets.

Keywords: Colubridae; evolution; proteins; snake; toxins; transcriptomics

1. Introduction
More than one hundred years of biochemical and pharmacological studies have resulted in
an exceptional depth of knowledge about snake venoms. The major toxins of the most medically
important taxa of venomous snakes were determined by first generation approaches including protein
chemistry, comparative pharmacology and cladistics methods borrowed from evolutionary biology.
Advances in molecular biology, particularly protein and nucleic acid sequencing techniques, greatly
expanded our understanding of compositional complexity, and more recent development in proteomics
and early genomics greatly accelerated the pace of cataloguing venoms in exquisite detail. Recent next
generation methods, including deep sequencing transcriptomics (RNAseq), genomic sequencing
and high resolution mass spectrometry, including top-down proteomics, generally called venomics;
(cf. [1–3]), have further accelerated the pace of sequence acquisition and compositional analysis and
constituted the basis of large-scale biotechnological explorative initiatives (e.g., [4]). These studies
collectively created very complete inventories of the toxin families and superfamilies present in

Toxins 2016, 8, 230; doi:10.3390/toxins8080230 www.mdpi.com/journal/toxins

Toxins 2016, 8, 230 2 of 24

species representing significant risk to human health, further refined by a growing knowledge of
the relative abundances, post-translational modifications and also structural conservation of proteins
across numerous genera [5–18]. As a side product of the accumulation of this knowledge, the observed
differences in venom composition among related taxa are becoming appreciated as a productive model
for making evolutionary inferences about diversifying selection. In turn, the association of quantified
toxins with empirically demonstrated activities have allowed predictions of functional and ecological
roles for the species that produce these toxins (e.g., [19–22]).
However, because most of these efforts were driven by anthropocentric interests in understanding
the mechanisms of actions of toxins causing severely debilitating effects, the major focus has been on
the species of medical relevance, which are confined to only three families of modern snakes (Viperidae,
Elapidae and Atractaspididae). As a consequence, a large part of the biodiversity of venom-producing
snakes was not systematically evaluated in the same way, leaving a gap in our knowledge of the
repertoire of toxins from other groups of venomous snakes, specifically those that do not typically
result in serious human envenomations [23].
The advanced snakes (Caenophidea, superfamily Colubroidea) include a diverse assemblage
of species with an evolutionary history of over 100 million years, most of which possess a venom
production system [24–27]. The family “Colubridae” formerly referred to any caenophidian snake
not included in the three medically important families of venomous snakes, and this assemblage,
though acknowledged to be a paraphyletic group, resisted systematic consensus for many
years [28–31]. More recently, several groups have reclassified the “Colubridae” into several families
and subfamilies [32–35], but a true consensus classification is still lacking. In a more formal definition,
a “colubrid” snake refers only to species belonging to the family Colubridae, which currently includes
the subfamilies Natricinae, Pseudoxenodontinae, Dipsadinae, Scaphiodontophiinae, Calamariinae,
Grayiinae and Colubrinae [34]. This family represents about 50% of the extant snake fauna (distributed
in more than 1800 species), many with very distinct habits and diversification of species within each
subfamily, and this classification scheme likely still masks considerable differentiation. Additional
rear-fanged species, accounting for approximately 361 species, have been allocated to the families
Homalopsidae and Lamprophiidae [34]; further, some authors consider Natricidae (approximately
226 species) and Dipsadidae (approximately 754 species) as distinct families [36,37]. Hereafter, we will
use a broader definition of the term “colubrid” to refer to any of the families in the above paraphyletic
group and not only to the family Colubridae sensu stricto, though the vast majority of data discussed
here come from this family.
In spite of the uncertainties in phylogenies, snakes in these families often possess one or more
enlarged rear teeth (opisthoglyph dentition) that are typically associated with a pair of Duvernoy’s
venom glands, homologs of the venom glands of families Viperidae, Elapidae and Atractaspididae [38,39].
Several species show no specialized or enlarged rear fangs (aglyph dentition), though in some cases
they also contain other specialized oral glands that produce venom-like secretions [40].
Colubrids are rarely investigated using -omics approaches mainly because of their limited capacity
to inject a debilitating dose of venom into humans, and so the biological activity of most species’
venoms is wholly unknown. As are all snakes, they are predators, and venom is presumed to be
of critical importance for capturing, killing and/or digesting the prey [41]. Thus, their venoms are
expected to be highly efficient within the proper ecological context of each species, meaning that
their venoms could be as rich and diverse in protein types as those from medically important species.
Moreover, because the different colubrid venoms are utilized in very distinct ecological scenarios
and evolved under different selective pressures, they may contain cryptic novel and unpredictable
types of proteins.
Because so few studies have focused on colubrids venoms and a plethora of different
methodological approaches were used by different labs, it is not clear which types of toxins
are currently known in the various groups, what structural characteristics are known and what
their evolutionary history has been. Some of the toxin sequences were obtained through direct
Toxins 2016, 8, 230 3 of 24

protein purification/sequencing, while others were deduced from transcriptomic and/or proteomic
investigations. In addition to the different times when the investigations were performed, their specific
goals sometimes hindered the perception of unusual new toxins. As a consequence, this has produced
a distorted view of toxin repertoires that exist in colubrid venoms and hinders a more complete
reconstruction of the evolutionary history of venom protein classes. The somewhat myopic view
of venoms as occurring only in front-fanged snake species has interfered with a more holistic,
fundamental perspective of the processes underpinning the evolution of venom, restricting the use of
these exceptionally diversified animals as models for testing adaptive evolution by natural selection
and negatively impacting the discovery of new bioactive molecules.
Here we survey previously discovered and several new venom proteins from venoms of colubrid
species, focusing on those with known protein or cDNA-based sequences. Our intent is to provide
an up-to-date catalog of proteins known to occur in colubrid snake venoms and present these
in an evolutionary context, highlighting their (presently known) diversification. There are well
over 2200 species of non-front-fanged snakes, many of which possess a Duvernoy’s venom gland,
so it should be immediately apparent that there is much work to do before a well-documented
understanding of venom diversity among colubrids is possible. Nonetheless, by summarizing known
information, we hope that this report will stimulate further investigation of the many genera of
colubrid snakes for which we have no toxinological information.

2. Results and Discussion

2.1. Compiling the Venom Components of Colubrid Snakes

Our attempt to compile the toxins present in colubrids was based on three strategies: (1) generating
transcriptomic sequences from the venom glands of three species of colubrids, Erythrolamprus miliaris,
Oxyrhopus guibei and Xenodon merremi (Dipsadinae subfamily of Colubridae), to identify transcripts
coding for known and putative types of snake toxins (Table S1); (2) prospecting public databases
for toxin-related sequences in other colubrid species previously investigated; and (3) reviewing the
literature on colubrid venoms that describes the isolation of toxins or provides clear evidence for the
occurrence of specific proteins in colubrid venoms. For ease of presentation, the protein types compiled
were organized into three categories: (a) “major snake venom components” (Table 1), referring to
protein types generally encountered in high amounts in the venoms of many species of traditionally
venomous snakes (Viperidae, Elapidae and Atractaspididae) and which certainly are important toxins;
(b) “minor (or arguably) venom components” (Table 2), referring to protein types previously described
in the venom of some species of venomous snakes, generally in low amounts, and which may represent
toxins, ancillary venom proteins or housekeeping proteins; and (c) “putative new snake toxins in
colubrid venoms” (Table 3), referring to protein types uncovered from colubrid venom analyses,
occurring in high or low quantities, which may represent putative toxins, exclusive or not to the group.
We should emphasize that the separation into major and minor components is unrelated to the level of
expression (or protein quantity) of the components in colubrid venoms. Rather, it is related to a relative
importance and frequency of the proteins in venoms of other venomous snakes. This organization is
admittedly subjective and flexible, but it was adopted because it would be unrealistic to propose a
division based on more tangible (but highly diverse) measures provided by the varied methodologies
adopted in the studies reviewed. Because it reflects a particular point of view, it does not aim to
establish a strict rule for toxin categorization or to define whether certain venom proteins do or do not
have relevant functions in snake venoms. Additionally, because the strict definition of “toxin” would
be dependent on the functional, ecological and behavioral contexts of the species, which are largely
unavailable for colubrids, the protein types included here should be generally regarded as “venom
components”, which in some cases are very likely to be toxins and in other cases may or may not be
toxins. The approximate phylogenetic relationships among the species for which venom components
could be identified in our compilation are depicted in trees (Figure 1) based on the phylogenetic
hypothesis of Colubroidea snakes as proposed by Pyron et al. [34].
Toxins 2016, 8, 230 4 of 24

Table 1. Major snake venom components and their occurrences in colubrid species.

Enzymatic Non-Enzymatic
Species Reference
LAAO PLA2 (IA) SVMP SVSP 3FTx CNP CRISP CTL DEFEN KUN-1 KUN-2
Boiga dendrophila B [42,43]
Boiga irregularis TPB TPB T TP T t [12,44,45]
Borikenophis portoricensis B BP [46,47]
Cerberus rynchops TP TP TP [48]
Coelognathus radiatus B [49]
Dispholidus typus xP x x [50–52]
Erythrolamprus miliaris T t T T This work; [50]
Erythrolamprus
x x x x [50,51]
poecilogyrus
Helicops angulatus BP [53]
Hypsiglena sp. TP T T TP TP t [13]
Hypsiglena torquata P [54]
Leoiheterodon
x x x [50]
madagascarensis
Macropisthodon rudis t [55]
Opheodrys aestivus x t t t [9]
Oxybelis fulgidus B [56]
Oxyrhopus guibei T t T T t This work
Phalotris mertensi TP T tP t t t T TP TP [57]
Pantherophis guttatus t x t t t [9]
Philodryas baroni P [54]
Philodryas chamissonis x x x x x [58]
Philodryas olfersii xTP xTP T xTP TP x [51,59]
Philodryas patagoniensis P [54]
Pseudoferania polylepis x x x x [50]
Rhabdophis tigrinus x x t [50]
Telescopus dhara x x x x [50,51]
Thamnodynastes strigatus TP t* t TP TP T [60]
Thrasops jacksonii x x x [51]
Trimorphodon biscutatus B B B [54,61]
Xenodon merremi T T T* T T This work
Protein categories are: LAAO, L-amino acid oxidase; PLA2 (IA), phospholipase A2 (type IA); SVMP, snake venom metalloproteinase; SVSP, snake venom serine proteinase;
3FTx, three finger toxin; CNP, C-type natriuretic peptide; CRISP, cysteine rich secretory protein; CTL, C-type lectin, DEFEN, defensin (crotamine-like); KUN-1, Kunitz type protein
(type 1); and KUN-2, Kunitz type protein (type 2). Types of evidence: T = Expressed in VG transcriptome at high level; t = Expressed in VG transcriptome at low (or uninformed) level;
x = RT-PCR (non-quantitative); P = Detected in the proteome by MS/MS; and B = Protein purified and/or activity tested from the Duvernoy’s venom. The green color graduation
represents the strength of the combination of evidence for each product, from light (less) to dark (more). Note: * = only 31 UTR detected.
Toxins 2016, 8, 230 5 of 24

Table 2. Minor snake venom components and their occurrences in colubrid species.

Enzymatic Non-Enzymatic
Species Reference
5NUCLEO
AChE DPP FactV FactX HYAL PDE AVIT bPLA2i CVF CYST gPLA2i KU-WA-FU NGF * OHA VEGF-A ** WAP
Boiga dendrophila [42,43]
Boiga irregularis T t t t t t t t t [12,44,45]
Borikenophis portoricensis B B [46,47]
Cerberus rynchops [48]
Coelognathus radiatus [49]
Dispholidus typus [50–52]
Erythrolamprus miliaris t t t t T t xt This work; [50]
Erythrolamprus poecilogyrus x [50,51]
Helicops angulatus [53]
Hypsiglena sp. t tP t t t [13]
Hypsiglena torquata [54]
Leoiheterodon madagascarensis [50]
Macropisthodon rudis t [55]
Opheodrys aestivus t t t x t x t t t [9]
Oxybelis fulgidus [56]
Oxyrhopus guibei t t t t t t This work
Phalotris mertensi tP t t t tP t tP t T [57]
Pantherophis guttatus t t *** t t *** t x t t t [9]
Philodryas baroni [54]
Philodryas chamissonis [58]
Philodryas olfersii t T x [51,59]
Philodryas patagoniensis [54]
Pseudoferania polylepis [50]
Rhabdophis tigrinus x [50]
Telescopus dhara [50,51]
Thamnodynastes strigatus t t [60]
Thrasops jacksonii x [51]
Trimorphodon biscutatus x [54,61]
Xenodon merremi t* This work
Protein categories are: 5NUCLEO, 51 nucleotidase; AChE, acetylcholinesterase; DPP, dipeptidyl peptidase; FactV, venom coagulation factor V; FactX, venom coagulation factor X;
HYAL, hyaluronidase; PDE, phosphodiesterase; AVIT, AVIT protein; bPLA2i, beta type phospholipase A2 inhibitor; CVF, cobra venom factor; CYST, cystatins; gPLA2i, gamma type
phospholipase A2 inhibitor; KU-WA-FU, ku-wap-fusin protein; NGF, nerve growth factor; OHA, ohanin (vesprin) protein; VEGF-A, vascular endothelial growth factor (type A);
and WAP, waprin-like proteins. Types of evidence: T = Expressed in VG transcriptome at high level; t = Expressed in VG transcriptome at low (or uninformed) level; x = RT-PCR
(non-quantitative); P = Detected in the proteome by MS/MS; and B = Protein purified and/or activity tested from the Duvernoy’s venom. The color gradation represents the strength of
the combination of evidence for each product, from light (less) to dark (more). Note: * partial sequences from other colubrids were PCR amplified as part of a phylogenetic study [62];
** no VEGF-F (svVEGF) detected in colubrids; *** cDNA and protein isolated from liver and serum of P. quadrivirgata and P. climacophora.
Toxins 2016, 8, 230 6 of 24

Table 3. Putative new snake venom components identified from colubrid species.

Enzymatic Non-Enzymatic
Species Reference
svLIPA PLA2 (IIE) PLB svMMP EGFr Lacta LIPO Vefico
Boiga dendrophila [42,43]
Boiga irregularis t t t t [12,44,45]
Borikenophis portoricensis [46,47]
Cerberus rynchops TP [48]
Coelognathus radiatus [49]
Dispholidus typus x x [50–52]
Erythrolamprus miliaris t T T This work; [50]
Erythrolamprus poecilogyrus x [50,51]
Helicops angulatus [53]
Hypsiglena sp. t [13]
Hypsiglena torquata [54]
Leoiheterodon madagascarensis x [50]
Macropisthodon rudis [55]
Opheodrys aestivus t t t [9]
Oxybelis fulgidus [56]
Oxyrhopus guibei t T t t T T t This work
Phalotris mertensi TP t tP t [57]
Pantherophis guttatus t t t [9]
Philodryas baroni [54]
Philodryas chamissonis [58]
Philodryas olfersii t T t t [51,59]
Philodryas patagoniensis [54]
Pseudoferania polylepis x [50]
Rhabdophis tigrinus TB x [50]
Telescopus dhara [50,51]
Thamnodynastes strigatus TP T TP [60]
Thrasops jacksonii [51]
Trimorphodon biscutatus x [54,61]
Xenodon merremi This work
Protein categories are: LIPA, snake venom acid lipase; PLA2 (IIE), phospholipase A2 (type IIE); PLB, phospholipase B; svMMP, snake venom matrix metalloproteinase; EGFr, EGF
repeats protein; Lacta, lactadherin-like protein; LIPO, lipocalin; and Vefico, veficolin (ficolin-like). Types of evidence: T = Expressed in VG transcriptome at high level; t = Expressed in
VG transcriptome at low (or uninformed) level; x = RT-PCR (non-quantitative); P = Detected in the proteome by MS/MS; and B = Protein purified and/or activity tested from the
Duvernoy’s venom. The color gradation represents the strength of the combination of evidence for each product, from light (less) to dark (more).
Toxins 2016, 8, 230 7 of 24
Toxins 2016, 8, 230  7 of 23 

 
Figure 1. Schematic cladograms showing the phylogenetic relationships among families and species 
Figure 1. Schematic cladograms showing the phylogenetic relationships among families and species of
of snakes discussed in this work (colored branches). The cladogram was based on the phylogenetic 
snakes discussed in this work (colored branches). The cladogram was based on the phylogenetic tree
tree proposed by Pyron et al. [34]. Dashed lines in Philodryas indicate the presumed placement of P. 
proposed by Pyron et al. [34]. Dashed lines in Philodryas indicate the presumed placement of P. chamissonis.
chamissonis. 

It is interesting to note that most -omics characterizations of colubrid venoms have addressed
It is interesting to note that most ‐omics characterizations of colubrid venoms have addressed 
members of the Dipsadinae subfamily of Colubridae, perhaps because a large number of genera
members of the Dipsadinae subfamily of Colubridae, perhaps because a large number of genera in 
in this 
this subfamily 
subfamily are 
are rear‐fanged 
rear-fangedand 
andpossess 
possessDuvernoy’s 
Duvernoy’svenom venomglands, 
glands,and 
andseveral 
several have
have  been
been 
involved
involved  in in 
human
human envenomations,
envenomations, typically
typically with
with  mild
mild  effects [63–65]. The 
effects  [63–65].  TheDipsadinae 
Dipsadinaespecies 
species
studied include Philodryas olfersii [59], Thamnodynastes strigatus [60] and Hypsiglena sp. [12], as well as
studied include Philodryas olfersii [59], Thamnodynastes strigatus [60] and Hypsiglena sp. [12], as well as 
Erythrolamprus 
Erythrolamprus miliaris, Oxyrhopus
miliaris, Oxyrhopus guibei
guibei  and
and  Xenodon 
Xenodon merremi 
merremidescribed 
describedhere. 
here.For 
ForColubrinae, 
Colubrinae,
transcriptomes of oral glands from Pantherophis guttatus, Opheodrys aestivus [9] and Boiga irregularis 
transcriptomes of oral glands from Pantherophis guttatus, Opheodrys aestivus [9] and Boiga irregularis
(Duvernoy’s venom gland); [12] were generated, although only the last one was complemented by 
(Duvernoy’s venom gland); [12] were generated, although only the last one was complemented by venom
venom  analysis.
proteomic proteomic  analysis.  Nevertheless, 
Nevertheless, many 
many toxins from thetoxins  from  the  other 
other subfamilies subfamilies 
have been have by
investigated been 
more
investigated  by  more  focused  approaches,  such  as  protein  purification  from  the  venom 
focused approaches, such as protein purification from the venom (e.g., Borikenophis portoricensis [47]) or (e.g., 
Borikenophis portoricensis [47]) or specific cDNA cloning, including some genera with particularly toxic 
specific cDNA cloning, including some genera with particularly toxic venom, such as the natricine
venom, such as the natricine Rhabdophis [66]. Very recently, full length mRNAs derived from secreted 
Rhabdophis [66]. Very recently, full length mRNAs derived from secreted venoms of several colubrine
venoms  of  several  colubrine  and  dipsadine  colubrids  were  reverse  transcribed  and  sequenced, 
and dipsadine colubrids were reverse transcribed and sequenced, demonstrating that it is possible to
demonstrating that it is possible to obtain transcript sequences from venom alone [67]. 
obtain transcript sequences from venom alone [67].
2.2. Major Snake Venom Enzymatic Components 
2.2. Major Snake Venom Enzymatic Components
For  most  colubrid  species,  especially  in  the  subfamily  Dipsadinae,  snake  venom 
For most colubrid species, especially in the subfamily Dipsadinae, snake venom metalloproteinases
metalloproteinases  (SVMPs)  are  predominant  components  in  the  transcriptomes  and  in  the 
(SVMPs) are predominant components in the transcriptomes and in the proteomes. All sequences
proteomes. All sequences described in Colubridae to date belong to the P‐III class of SVMPs, which 
described in Colubridae to date belong to the P-III class of SVMPs, which include pre- and pro-domains,
include pre‐ and pro‐domains, a metalloproteinase catalytic domain, a disintegrin‐like domain and a 
a metalloproteinase catalytic domain, a disintegrin-like domain and a cysteine-rich domain (Figure 2).
cysteine‐rich domain (Figure 2). The absence of P‐II, P‐I and short coding disintegrins in colubrid 
The absence of P-II, P-I and short coding disintegrins in colubrid venoms is in accordance with the
venoms is in accordance with the hypothesis that those proteins evolved within the family Viperidae 
hypothesis that those proteins evolved within the family Viperidae from a P-III ancestor gene, after the
from a P‐III ancestor gene, after the split of this lineage [68,69]. A solely exception in Colubridae is 
split of this lineage [68,69]. A solely exception in Colubridae is the occurrence of a shortened P-III
Toxins 2016, 8, 230
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8 of 23 

the  occurrence  of  a  shortened  P‐III  SVMP  in  Phalotris  mertensi.  This  protein  was  proteomically 
SVMP in Phalotris mertensi. This protein was proteomically confirmed in the venom of the species and
confirmed in the venom of the species and it has a partial disintegrin‐like domain and no Cys‐rich 
it has a partial disintegrin-like domain and no Cys-rich domain, as a result of a transcript with an
domain, as a result of a transcript with an early stop codon and a substituted 3′UTR sequence [57]. A 
early stop codon and a substituted 31 UTR sequence [57]. A phylogenetic tree of representative SVMPs
phylogenetic tree of representative SVMPs indicates that, despite a high degree of diversity among 
indicates that, despite a high degree of diversity among the Colubridae SVMPs, they share a common
the Colubridae SVMPs, they share a common ancestor with elapid and atractaspidid P‐III SVMPs 
ancestor with elapid and atractaspidid P-III SVMPs (Figure 2).
(Figure 2). 

 
Figure  2.  Maximum  likelihood  tree  showing  the  relationship  among  representative  SVMPs  from 
Figure 2. Maximum likelihood tree showing the relationship among representative SVMPs from
different snake families. Bootstrap values are plotted close to the internal nodes. Colors in the terminal 
different snake families. Bootstrap values are plotted close to the internal nodes. Colors in the
nodes indicate the types of the precursors, and their domain arrangements are depicted on the right. 
terminal nodes indicate the types of the precursors, and their domain arrangements are depicted on
Abbreviated  domains  are:  S,  signal  peptide;  Pro,  prodomain;  Catalytic,  metalloproteinase;  D‐like, 
the right. Abbreviated domains are: S, signal peptide; Pro, prodomain; Catalytic, metalloproteinase;
disintegrin‐like;  Dis,  disintegrin;  Cys,  cysteine  rich;  TM,  transmembrane;  EGF,  epidermal  growth 
D-like, disintegrin-like; Dis, disintegrin; Cys, cysteine rich; TM, transmembrane; EGF, epidermal
factor; and Cytopl, cytoplasmic. The protein sequences are referred to by their accession numbers in 
growth factor; and Cytopl, cytoplasmic. The protein sequences are referred to by their accession
GenBank,  except  those  initiated  by  the  codes  EMILISO,  OGUIISO,  PMERREF,  TSTRCLU  and 
numbers in GenBank, except those initiated by the codes EMILISO, OGUIISO, PMERREF, TSTRCLU
XMERCLU,  which  are  mentioned  in  the  “definition”  field  of  sequence  files  deposited  in  the 
and XMERCLU, which are mentioned in the “definition” field of sequence files deposited in the
Transcriptome Shotgun Assembly (TSA) database. 
Transcriptome Shotgun Assembly (TSA) database.
Snake  venom  serine  proteinases  (SVSPs)  are  detected  in  some  colubrid  venoms  and 
transcriptomes; 
Snake venomhowever, 
serine they  are  not  (SVSPs)
proteinases commonly  present 
are in  these 
detected venoms, 
in some nor  as  venoms
colubrid abundantly and
expressed and diversified as observed in many viperid snakes. The few colubrid SVSPs sequenced 
transcriptomes; however, they are not commonly present in these venoms, nor as abundantly expressed
andare related to the kallikrein‐like enzymes well characterized in viperid venoms, and they include a 
diversified as observed in many viperid snakes. The few colubrid SVSPs sequenced are related
C‐terminal extension that distinguishes them from the lizard venom kallikrein‐like enzymes [70]. 
to the kallikrein-like enzymes well characterized in viperid venoms, and they include a C-terminal  
Phospholipases  A 2  (PLA2)  are  very  common  components  in  the  venoms  of  the  medically 
extension that distinguishes them from the lizard venom kallikrein-like enzymes [70].
important snake families Elapidae and Viperidae, and they belong to type I and II PLA
Phospholipases A2 (PLA2 ) are very common components in the venoms of 2s, respectively. 
the medically
important snake families Elapidae and Viperidae, and they belong to type I and II PLA2 s, respectively.
Toxins 2016, 8, 230 9 of 24

In colubrids, they seem to not be among major components and have been detected in only a few
species. In the colubrine Trimorphodon biscutatus, an enzyme was purified and its partial sequence
indicated that it was a type IA PLA2 [61]. However, in another colubrine (Dispholidus typus) [51],
in the dipsadine Oxyrhopus guibei (this work) and in the pseudoxyrhophiine (family Lamprophiidae)
Leioheterodon madagascariensis [51], among others, the reported type of PLA2 is IIE.
The occurrence of transcripts coding for enzymes of IIE subtype in the venom glands indicates a
possible independent recruitment of a PLA2 to the venom, since they are distinct from the type IIA
paralogs commonly expressed in the venom glands of viperid snakes [51]. Whether or not these type
IIE PLA2 s represent truly new toxins or accessory proteins of the venom glands remains to be clarified,
but Hargreaves et al. [9] found them to be exclusively expressed at low levels in the venom glands of
the species tested.
Despite being very common in the venom of other groups of snakes, L-Amino Acid Oxidase
(LAAO) was thought to be essentially non-existent in colubrid venoms (e.g., [71]). Very low levels
of LAAO activity were detected in Brown Treesnake (B. irregularis) venom [72]; however, assays of
venom from 13 different species of colubrine, dipsadine and natricine rear-fanged snakes detected
no LAAO activity [73]. In a comparative transcriptomic analysis of tissues from Pantherophis guttatus,
an LAAO was shown to be expressed in the scent gland but not in the salivary glands of this species [9],
suggesting it is not a venom component. Recently, however, an LAAO was found moderately expressed
in the venom glands of the colubrid Phalotris mertensi, and the MS/MS spectrometric analysis clearly
showed its presence in the venom of this species [57].

2.3. Major Snake Venom Non-Enzymatic Components

Three-Finger Toxins (3FTx) are major constituents of Elapidae venoms and represent the lethal
component of the majority of species of this family. These toxins seem to have differential importance
in different subfamilies of colubrids. Alpha-colubritoxin from Coelognathus radiatus was the first
colubrid toxin isolated and sequenced [49] and several other 3FTx, such as denmotoxin and irditoxin,
functionally characterized in members of the subfamily Colubrinae, were demonstrated to be abundant
toxins with taxon-specific activities [42,45]. The -omics characterization of Boiga irregularis venom
showed that 3FTx dominate the transcriptome of this species (67.5% of toxin transcripts) [12].
The authors described 58 unique 3FTx sequences grouped into at least 10 sequence clusters that
were proteomically confirmed in the venom. These clusters could be arranged in three groups based on
the structural characteristics, but none of them were closely related to the above-mentioned irditoxin
from the same species. Together with individual sequences isolated from other genera [9,50,56],
3FTx seem to be major components in many venoms of the subfamily Colubrinae. In contrast, in the
Dipsadinae, 3FTx are not found [59,60] or are detected at minor abundance and diversity levels [57].
However, in the current work, we retrieved sequences from the transcriptome of the Duvernoy’s
venom gland of Xenodon merremi (a dipsadine colubrid) that were expressed at high level (Table 1).
3FTx-like sequences were also reported in venom glands of the family Lamprophiidae, as well as in
species at the base of the Alethinophidia snake radiation (including species in the Cylindrophiidae
and Pythonidae [50]). Nevertheless, it appears likely that 3FTx-like transcripts found in gland tissue of
these latter two families may represent house-keeping genes, rather than toxins [9,74].
C-Type Lectins (CTL) are ubiquitous venom components in many snake groups, and they are also
found abundantly in colubrid venoms. In Colubridae, amounts of venom CTL transcripts vary from 2%
in Philodryas olfersii [59] to as much as 21% in Phalotris mertensi of total transcripts [57]. They were also
reported in the snakes Pseudoferania polylepis and Cerberus rynchops (family Homalopsidae) and were
highly expressed in Cerberus [48]. From a phylogenetic tree of all colubrid CTLs and related orthologs
(Figure 3), it is possible to observe the existence of distinct types of CTLs in non-front fanged snakes,
although the phylogenetic reconstruction failed to resolve the evolutionary relationships among them.
Nevertheless, in addition to the presence of a CTL-like (snalec) clade, largely found in colubrids,
colubrid sequences are observed to be nested within the clade of Elapidae and Viperidae “true” CTLs
Toxins 2016, 8, 230 10 of 24

sequences (i.e., those

Toxins 2016, 8, 230  with a predicted galactose binding motif QPD substituting the plesiotypic10 of 23  motif
EPN: [75,76]. One of them (PMERREF_CTL04) was confirmed in P. mertensi venom and in fact has
mertensi 
the QPDvenom  and  in  fact that
motif, indicating has predicted
the  QPD  motif,  indicating  that 
galactose-binding predicted 
lectins shouldgalactose‐binding 
also be present inlectins 
other
should also be present in other Colubridae venoms. Moreover, some orphan transcripts observed in 
Colubridae venoms. Moreover, some orphan transcripts observed in the venom glands of snakes
the 
fromvenom  glands 
different of  snakes 
families from 
clustered different  families 
completely outside ofclustered 
the cladescompletely 
of typicaloutside 
venom of  the (Figure
CTLs clades  3).
of 
typical venom CTLs (Figure 3). Some of them are suggested to code for venom proteins, such as two 
Some of them are suggested to code for venom proteins, such as two transcripts highly expressed and
transcripts 
proteomicallyhighly  expressed 
detected and  proteomically 
in the venom detected 
of P. mertensi, in  the 
and similar venom  of 
transcripts areP. expressed
mertensi,  at
and  similar 
moderate
transcripts are expressed at moderate levels in the venom glands of other colubrids (Hypsiglena sp. 
levels in the venom glands of other colubrids (Hypsiglena sp. and Boiga irregularis; [12]), viperids
and Boiga irregularis; [12]), viperids (Bothrops insularis; [77]) and elapids (Hoplocephalus bungaroides; 
(Bothrops insularis; [77]) and elapids (Hoplocephalus bungaroides; [78]). Interestingly, the encoded proteins
[78]). Interestingly, the encoded proteins from the transcripts of this group present not a single but 
from the transcripts of this group present not a single but various motifs (QPD, EPD, EPN, RPS, QVE,
various motifs (QPD, EPD, EPN, RPS, QVE, and EPK) for sugar binding at the second loop of the 
and EPK) for sugar binding at the second loop of the carbohydrate recognition domain. It indicates
carbohydrate recognition domain. It indicates that these genes may have undergone a diversification 
that these genes may have undergone a diversification process that parallels that experienced by other
process that parallels that experienced by other CTL types, i.e., the substitution of the binding motif 
CTL types, i.e., the substitution of the binding motif of the original sugar ligand, mannose, by binding
of the original sugar ligand, mannose, by binding motifs to other types of carbohydrates. 
motifs to other types of carbohydrates.  

 
Figure  3.  Maximum 
Figure 3. Maximum likelihood 
likelihood circular 
circular cladogram 
cladogram showing 
showing the  relationship  among
the relationship among  representative
representative 
CTLs from different snake families. Bootstrap values are plotted close to internal nodes. Colors at the 
CTLs from different snake families. Bootstrap values are plotted close to internal nodes. Colors at the
terminal nodes (circles) indicate typical vs. atypical venom proteins and the evidence of occurrence 
terminal nodes (circles) indicate typical vs. atypical venom proteins and the evidence of occurrence
in the venoms. Colors in the diagram surrounding the cladogram indicate the taxonomic groups. The 
in the venoms. Colors in the diagram surrounding the cladogram indicate the taxonomic groups.
carbohydrate binding motifs, as discussed in the text (EPN, QPD, etc.), are indicated by red type. The 
The carbohydrate binding motifs, as discussed in the text (EPN, QPD, etc.), are indicated by red type.
protein  sequences 
The protein are are
sequences referred  by by
referred their  GenBank 
their accession 
GenBank accessionnumbers, 
numbers,except 
exceptthose 
thoseinitiated 
initiated by 
by the 
the
codes EMILISO, OGUIISO, PMERREF and XMERCLU, which are mentioned in the “definition” field 
codes EMILISO, OGUIISO, PMERREF and XMERCLU, which are mentioned in the “definition” field
of sequence files deposited in TSA. 
of sequence files deposited in TSA.

Although the role of cysteine rich secretory proteins (CRISPs) in venom is not yet clear, they are 
very ubiquitous venom components and are found in almost all snake species, including colubrids, 
and have been investigated via either classical protein techniques (e.g., [79]) or ‐omics profiling [80]. 
Contrary to the other highly expressed snake toxins, CRISPs seem to have not undergone multiple 
Toxins 2016, 8, 230 11 of 24

Although the role of cysteine rich secretory proteins (CRISPs) in venom is not yet clear, they are
very ubiquitous venom components and are found in almost all snake species, including colubrids,
and have been investigated via either classical protein techniques (e.g., [79]) or -omics profiling [80].
Contrary to the other highly expressed snake toxins, CRISPs seem to have not undergone multiple
Toxins 2016, 8, 230  11 of 23 
duplications during snake lineage evolution, and a single paralog is normally found abundantly
duplications 
expressed and during 
translatedsnake 
to lineage 
a venom evolution, 
protein and  a  single 
in each paralog 
colubrid is  normally 
species; in some found  abundantly 
species, such as
expressed and 
B. irregularis, translated 
a minor isoformto  a isvenom  protein in 
also present each 
in the colubrid 
venom species; in some 
(Mackessy, unpub. species, such  as B. 
obs.). Nevertheless,
irregularis,  a  minor  isoform  is  also  present  in  the  venom  (Mackessy, 
positive Darwinian selection on CRISPs were observed to be higher in Colubridae and Viperidae unpub.  obs.).  Nevertheless, 
positive 
proteins thanDarwinian 
on otherselection  on  CRISPs 
reptiles, while were 
negative observed 
selection to  be in
occurs higher  in  Colubridae 
mammalian CRISPsand  Viperidae 
[80].
proteins than on other reptiles, while negative selection occurs in mammalian CRISPs [80]. 
The first C-type natriuretic precursor (CNP) from a colubrid species was described  from the
The 
P. olfersii first  C‐type  natriuretic 
transcriptome, where it precursor  (CNP) to
was suggested from 
havea  colubrid 
a common species  was  described 
ancestor with the from  the   
natriuretic
P.  olfersii  transcriptome,  where  it  was  suggested  to  have  a  common  ancestor  with  the  natriuretic 
peptide precursor of elapid snakes and with the bradykinin-potentiating peptides precursor (BPP)
peptide precursor of elapid snakes and with the bradykinin‐potentiating peptides precursor (BPP) of 
of viperid snakes [59]. Currently, nine colubrid species in the three major subfamilies of Colubridae
viperid  snakes  [59].  Currently,  nine  colubrid  species  in  the  three  major  subfamilies  of  Colubridae 
(Colubrinae, Dipsadinae and Natricinae) were shown to have this precursor generally highly expressed
(Colubrinae,  Dipsadinae  and  Natricinae)  were  shown  to  have  this  precursor  generally  highly 
in the Duvernoy’s venom glands. Most of them have the same general structure, i.e., the C-type peptide
expressed in the Duvernoy’s venom glands. Most of them have the same general structure, i.e., the 
has no C-terminal extension and the CNP prodomain is not preceded by a BPP-containing region
C‐type  peptide  has  no  C‐terminal  extension  and  the  CNP  prodomain  is  not  preceded  by  a  BPP‐
(Figure 4). Based
containing  on this
region  organization,
(Figure  4).  Based  Jackson etorganization, 
on  this  al. [78] suggested that et 
Jackson  theal. 
acquisition of the C-terminal
[78]  suggested  that  the 
extension occurred within the Elapidae, while the acquisition of BBP repeats
acquisition of the C‐terminal extension occurred within the Elapidae, while the acquisition of BBP  occurred along the viperid
lineage diversification. We notice, however, a notable exception in the CNP precursor of the Dipsadinae
repeats occurred along the viperid lineage diversification. We notice, however, a notable exception 
P. mertensi: this precursor, transcribed at high levels in the venom glands, possesses a long sequence
in the CNP precursor of the Dipsadinae P. mertensi: this precursor, transcribed at high levels in the 
inserted
venom  at glands, 
the middle of the CNP
possesses  prodomain
a  long  (linker
sequence  domain),
inserted  which
at  the  is richof inthe 
middle  Pro CNP 
residues (including
prodomain   
PP(linker domain), which is rich in Pro residues (including PP and PPP internal peptides) and resembles 
and PPP internal peptides) and resembles the BPP-containing region of the viperid precursor
(Figure 4). At the C-terminal portion of this region, one particular motif, “QRFFPPPIPP”, shows a high
the BPP‐containing region of the viperid precursor (Figure 4). At the C‐terminal portion of this region, 
one particular motif, “QRFFPPPIPP”, shows a high degree of similarity to the BPP signature. Besides 
degree of similarity to the BPP signature. Besides the classical BPPs, which led to the development of
the classical BPPs, which led to the development of successful anti‐hypertensive drugs [81], the BPP 
successful anti-hypertensive drugs [81], the BPP precursors of Viperidae snakes were demonstrated to
precursors of Viperidae snakes were demonstrated to generate other bioactive peptides, including 
generate other bioactive peptides, including SVMP inhibitors [82–85]. It is thus reasonable to suppose
thatSVMP  inhibitors 
this region [82–85]. 
of the It  is CNP
P. mertensi thus  precursor
reasonable could
to  suppose 
also bethat  this  region 
processed of  the  P. 
to generate mertensi 
bioactive CNP 
peptides
precursor could also be processed to generate bioactive peptides and perhaps a BPP‐like peptide. 
and perhaps a BPP-like peptide.  

 
Figure 4. Schematic organization of CNP (and BPP) precursors in the different snake families and in 
Figure 4. Schematic organization of CNP (and BPP) precursors in the different snake families and in
other vertebrates. The precursor of P. mertensi [57] exhibits a Pro‐rich insertion in the linker region 
other vertebrates. The precursor of P. mertensi [57] exhibits a Pro-rich insertion in the linker region
(detached at the bottom), which includes a BPP‐like segment that may generate a BPP after processing. 
(detached at the bottom), which includes a BPP-like segment that may generate a BPP after processing.

Crotamine  is  a  beta‐defensin‐type  polypeptide  very  well  characterized  from  rattlesnake 

Crotamine is a beta-defensin-type polypeptide very well characterized from rattlesnake (Viperidae)
(Viperidae)  venoms  and  thought  to  be  restricted  to  the  genus  Crotalus.  However,  beta‐defensin 
venoms and thought to be restricted to the genus Crotalus. However, beta-defensin homologous genes
homologous  genes  were  found  in  other  viperid  genera  [86]  and,  more  recently,  venom  gland 
were found in other viperid genera [86] and, more recently, venom gland transcripts were reported at
transcripts were reported at relatively high expression levels in the transcriptome of the colubrids 
relatively high expression levels in the transcriptome of the colubrids Thamnodynastes strigatus [60]
Thamnodynastes strigatus [60] and Phalotris mertensi [57]. In the latter, the corresponding protein was 
detected  by  shotgun  MS/MS  analysis  of  the  venom,  suggesting  it  may  be  a  valid  colubrid  venom 
component. The colubrid proteins have a highly conserved signal peptide, almost identical to that of 
crotamine (see Supporting Figure 3 from [60]); the mature polypeptides display the same cysteines 
Toxins 2016, 8, 230 12 of 24

and Phalotris mertensi [57]. In the latter, the corresponding protein was detected by shotgun MS/MS
analysis of the venom, suggesting it may be a valid colubrid venom component. The colubrid proteins
have a highly conserved signal peptide, almost identical to that of crotamine (see Supporting Figure 3
from [60]); the mature polypeptides display the same cysteines involved in the disulfide arrangement
of crotamine, but the other residues are highly variable, making it difficult to establish the evolutionary
relationship between them.
Kunitz-type proteins appear in snake venoms in several forms, sometimes as single-product
precursors (KUN-1), at other times with tandem repeated domains (KUN-2), and less frequently
associated with WAP domains in a protein designated ku-wap-fusin (KU-WA-FU) [87]. Although in
some species of colubrids these components have a transcriptional level not indicative of a relevant
participant in venom, in at least two species, Hypsiglena sp. [12] and Phalotris mertensi [57], they have
medium or elevated expression levels and were also detected in the venom. In Phalotris mertensi,
three single-domain precursors are highly expressed and dominate the venom profile. The conservation
of residues believed to be the protease inhibitory sites in their sequences [88,89] indicate they likely
act as serine proteinase inhibitors, the plesiotypic function of this toxin, rather than as neurotoxins, as
observed in some elapid Kunitz-like proteins.

2.4. Minor or Arguably Actual Venom Components

Other protein components previously reported in the venoms of the families Elapidae and/or
Viperidae, generally in minor quantities, are also detected in low amounts in colubrid venoms and/or
transcriptomes and are listed in Table 2. However, the actual contribution of these molecules to
the venom is debatable, and some authors consider them non-toxins because of their occurrence in
non-venom gland specific tissues [9].
Regarding minor occurring enzymes, venom-like acetylcholinesterase (Ache) sequences are found
in many elapid species but were suggested as a colubrid venom component only in Boiga venom,
where low activity was detected [44,73], as well as low expression levels in the transcriptome of
B. irregularis [12]. A 51 -nucleotidase, on the other hand, was identified at low expression levels in the
Phalotris mertensi transcriptome, and it was also detected in its venom proteome [57]. Factor Va- and
Factor Xa-like proteins are venom serine proteinases distinct from the classical SVSPs [90], and they are
believed to have been recruited into the venom proteome on the basis of their occurrences in venoms
of the Australian elapid radiation [50]. Accordingly, no Factor Va-like sequence were retrieved in our
searches, while only the endophysiological (non-venom) Factor Xa transcripts could be found in the
venom glands of three colubrid species but were never identified in the secreted venom. These data
indicate that the expression of endophysiological Factor Xa also may occur in the venom glands,
in addition to the liver, although at low levels (this work and [9]). Transcripts for some other minor
venom enzymes were only found at very low levels.
Growth factor sequences from colubrids and other snakes, such as vascular endothelial growth
factor (VEGF) and nerve growth factor (NGF), are common in the databases. However, there is no clear
evidence of them as venom components among colubrids. Whereas a venom-specific VEGF (VEGF-F)
was extensively demonstrated to be a venom component in viperid snakes, possibly acting as a toxin
dispersion agent [91], an endophysiological paralog (VEGF-A) was later shown to be co-expressed
in venom glands and secreted only in low amounts in the venom of some species [92]. All the VEGF
forms retrieved from colubrids are similar to VEGF-A, expressed at low levels, and thus they are
more likely to be non-toxins, possibly corresponding to the endophysiological factor produced in the
venom gland environment. The colubrid NGF sequences available in databases are mostly derived
from phylogenetic studies based on this genetic marker. In contrast to other venomous snake families,
where NGF was clearly demonstrated to be a venom component, in colubrids there is no support for
this factor as a venom component, since it is not specifically transcribed in the venom glands of any
species and the protein has to date not been isolated from the venom.
Toxins 2016, 8, 230 13 of 24

Cobra venom factor (CVF) was clearly demonstrated as a venom component only in elapid
snakes [93]. Although very similar sequences could be found expressed at low levels in some
colubrids, the absence of protein detection in their venom suggests the transcripts could also be
the endophysiological complement factor C3 expressed by blood cells within the venom glands.
Enzymatic inhibitors that typically function to protect snakes from the bites of other snakes
are mainly produced in the liver and secreted into the plasma of venomous and non-venomous
snakes [94], but some of them seem to be produced in the venom glands. For example, a specific
paralog of a gamma-PLA2 inhibitor (gPLA2 i) was shown to be exclusively expressed in venom glands
of B. jararaca (Viperidae) [10]. Accordingly, we could identify three colubrid species showing low
to medium expression levels of gPLA2 i, and one of them was proteomically demonstrated in the
venom. Protease inhibitors such as cystatins have been previous demonstrated in snake venoms [95],
but their role in the venom is unclear. We retrieved transcripts coding for these proteins from some
colubrids, but according to the analysis of Hargreaves et al. [9], they have undifferentiated levels
of expression among tissues, and no further evidence of their presence in colubrid venoms have
been noted yet, indicating that they are probably not colubrid venom components. Nevertheless, the
common occurrence of many transcripts coding for all these toxin-like proteins in venom-producing
tissues indicate that if they are not toxins, they may play important roles in the maintenance of
this specialized secretory epithelium. We did not find transcripts related to sarafotoxins [96] in any
colubrids, including Leioheterodon madagascariensis (Lamprophiidae), indicating that this component
may be apotypic of Atractaspidinae.

2.5. Putative New Snake Toxins Suggested from Colubrid Venoms

Although SVMPs dominate many colubrid venom profiles, another type of metzincin, the snake
venom matrix metalloproteinase (svMMP), was revealed to be a colubrid-specific venom component
likely playing an important role in some species. svMMPs were abundantly found in the transcriptome
and proteome of Thamnodynastes strigatus [60] and are highly expressed in the transcriptome of
Erythrolamprus miliaris (this work), both Dipsadinae. Other Dipsadinae species investigated by
similar -omics approaches showed lower abundance of svMMPs, though they were still detected
in the venoms of some species. A svMMP was also purified, sequenced and functionally characterized
from the venom of Rhabdophis tigrinus (Natricinae) [66]. For many of the species in which svMMPs
were detected, SVMPs were also present in the venom, seemingly indicating that svMMPs are not
substituting for the function of SVMPs but perhaps are adding a possible synergistic effect toward
producing extracellular matrix lesions caused by the venoms. The colubrid svMMPs show important
differences related to the presence of ancillary domains, as illustrated in Figure 5: whereas in some
species, such as Rhabdophis tigrinus, the protein has a classical MMP9-like structure, in others, such
as Thamnodynastes strigatus, they do not include the fibronectin repeats nor the hemopexin domains,
thus resembling a MMP7-like arrangement. The E. miliaris svMMPs found in this work revealed a
more complex situation, since some of the precursors have the fibronectin repeats inserted in the
catalytic domain, whereas other precursors do not show these domains (Figure 5). Both forms are
highly transcribed in the venom glands, representing the major toxin type found in the transcriptome
of this species, but unfortunately, we did not have access to the venom of this species to evaluate its
effective secretion. It is interesting to observe in a phylogenetic tree of svMMP precursors (Figure 5)
that there is a strong clustering of svMMPs within the MMP-9 clade. This result indicates that all
svMMPs seem to derive from a single MMP-9 ancestor gene, regardless of the presence or the absence
of ancillary domains. Additionally, the clustering of species-specific proteins in monophyletic groups
signifies intra-clade gene duplications, with independent losses of the fibronectin and hemopexin
domains in some clades (Thamnodynastes and Erythrolamprus). Moreover, it clearly points out that the
simplified MMP7-like arrangement observed in some svMMPs is a derived trait from the modification
of a MMP9-type svMMP, rather than originating from an MMP7 gene.
Toxins 2016, 8, 230  14 of 23 

monophyletic group, and thus LIPA may represent a novel type of venom component, and perhaps 
Toxins 2016, 8, 230 14 of 24
a toxin [57]. 

 
Figure 5. Maximum likelihood tree showing the relationship among svMMPs from different snake 
Figure 5. Maximum likelihood tree showing the relationship among svMMPs from different snake
families and MMPs from other vertebrate groups. Bootstrap values are plotted close to internal nodes. 
families and MMPs from other vertebrate groups. Bootstrap values are plotted close to internal nodes.
The domain arrangement of each precursor type is depicted on the right. The types of evidence for the
The domain arrangement of each precursor type is depicted on the right. The types of evidence for 
occurrence in venoms are indicated by “T” (transcribed) and “V” (detected in venom). The protein
the occurrence in venoms are indicated by “T” (transcribed) and “V” (detected in venom). The protein 
sequences are labeled by their accession numbers in GenBank, except those initiated by the codes
sequences  are  labeled  by  their  accession  numbers  in  GenBank,  except  those  initiated  by  the  codes 
EMILREF, OGUIISO, and PMERREF, which are mentioned in the “definition” field of sequence files
EMILREF, OGUIISO, and PMERREF, which are mentioned in the “definition” field of sequence files 
deposited in TSA.
deposited in TSA. 

Novel  non‐enzymatic  components  were  also  proposed  from  the  venoms  of  non‐front  fanged 
snakes.  Venom  ficolin  (veficolin)  is  a  class  of  putative  toxins  initially  characterized  from  the 
homalopsid  Cerberus  rynchops  venom  and  transcriptome  [48].  Other  related  transcripts  could  be 
retrieved  from  several  colubrid  species  but  they  are  generally  expressed  at  low  levels,  and  the 
encoded proteins were not detected in any other venom. A lactadherin‐like protein, a secreted carrier 
protein containing a FA58C (coagulation factor V and VIII C‐terminal) domain, was first identified 
from a partial clone in the transcriptome of T. strigatus. Since it was found proteomically in the venom 
of  that  species,  it  was  suggested  as  a  possible  venom  component  [60].  In  the  present  work,  we 
Toxins 2016, 8, 230 15 of 24

Another enzyme representing an example of a putative toxin from colubrid venom is an acid lipase
(svLIPA), similar to mammalian lysosomal acid lipases. In P. mertensi, this protein was proteomically
and immunochemically detected in the venom and its mRNA was highly expressed in the venom
glands [57]. Interestingly, this P. mertensi sequence is closely related to acid lipases previously suggested
as possible venom components in species of other snake families but not clearly demonstrated in their
venoms [97,98], as well as a prominent protein component of saliva from several species of Varanus
(BLAST search). By comparing acid lipase sequences from different reptiles, we could demonstrate
that all transcripts showing evidence for venom proteins in different snakes (i.e., high expression in
the venom glands, proteomics detection, or immunoreactivity in venom) form a monophyletic group,
and thus LIPA may represent a novel type of venom component, and perhaps a toxin [57].
Novel non-enzymatic components were also proposed from the venoms of non-front fanged
snakes. Venom ficolin (veficolin) is a class of putative toxins initially characterized from the homalopsid
Cerberus rynchops venom and transcriptome [48]. Other related transcripts could be retrieved from
several colubrid species but they are generally expressed at low levels, and the encoded proteins were
not detected in any other venom. A lactadherin-like protein, a secreted carrier protein containing a
FA58C (coagulation factor V and VIII C-terminal) domain, was first identified from a partial clone
in the transcriptome of T. strigatus. Since it was found proteomically in the venom of that species,
it was suggested as a possible venom component [60]. In the present work, we identified a complete
transcript coding for this protein in the Oxyrhopus guibei transcriptome, but we did not evaluate the
venom of this species. A search for similar transcripts in other snakes revealed a complete sequence
only in the transcriptome of the viperid Crotalus horridus (JAA96713, [15]). An EGF repeat-containing
cDNA was found in relatively high levels in the transcriptome of T. strigatus but was not confirmed in
this venom nor was it retrieved from other species [60].
An interesting case of a potentially new venom component identified from Colubridae -omics
analysis is a type of lipocalin. Transcripts coding for lipocalin-structured proteins were retrieved from
several snake venom glands by transcriptomic analysis or by RT-PCR amplification and they were
shown to be homologous [51]. In the transcriptomic analysis of the Atractaspidinae Atractaspis aterrima,
some lipocalin sequences were identified as among the most expressed transcripts in the venom
glands [18]. Since lipocalins are common components from some invertebrate venoms and from the
saliva of hematophagous animals [99], they were suggested as possible venom components [47].
These proteins also show weak sequence similarities to a putative olfactory protein specifically
expressed in high amounts in the Bowman’s glands of the olfactory tissue from a frog [100].
Interestingly, among the original data generated in the present work, we found an extremely highly
expressed transcript coding for a lipocalin in Oxyrhopus guibei. Alone, this mRNA accounts for 29%
of the sequencing reads in the transcriptomic analysis. A phylogenetic tree of all available lipocalin
sequences, from snakes and from several other sources, showed that the transcripts highly expressed in
snake venom glands, including those from Colubridae and Atractaspidinae, are likely to be orthologs,
whereas other transcripts expressed at low levels correspond to a paralogous snake gene (Figure 6).
Although it is not possible to confirm, without a proteomic analysis, if lipocalin is indeed a venom
component, the high expression of the same gene in the venom glands of distinct snake species suggests
that its product should have an important role for this animal, perhaps as a new toxin or perhaps
involved in olfactory-mediated behavior.
Finally, a putative new toxin proposed from a highly expressed transcript from Atractaspsis
aterrima (Atractaspidine) [18] displayed some similarity with an unknown protein predicted from a
high expressed contig from Erythrolamprus miliaris. However, the areas of conservation were restricted
to the signal peptide and to the C-terminal and thus it is not likely that the two putative proteins
correspond to a common toxin (data not shown).
in  snake  venom  glands,  including  those  from  Colubridae  and  Atractaspidinae,  are  likely  to  be 
orthologs, whereas other transcripts expressed at low levels correspond to a paralogous snake gene 
(Figure 6). Although it is not possible to confirm, without a proteomic analysis, if lipocalin is indeed 
a  venom  component,  the  high  expression  of  the  same  gene in  the  venom  glands  of distinct  snake 
species suggests that its product should have an important role for this animal, perhaps as a new 
Toxins 2016, 8, 230 16 of 24
toxin or perhaps involved in olfactory‐mediated behavior. 

 
Figure 6. Maximum likelihood tree showing the relationship among lipocalin proteins from different
snake families and from other vertebrate groups. Note that transcripts highly expressed in venom
glands are all in the same clade. Bootstrap values are plotted close to internal nodes. The protein
sequences are labeled by their accession numbers in GenBank, except those initiated by the code EMIL,
which is mentioned in the “definition” field of sequence files deposited in TSA and the sequence
OGUIREF_Lipo1 (Accession Number KX450875). Sequences labeled GAMF from A aterrima were
translated from the original nucleotide contigs retrieved from TSA.

3. Conclusions
It is now abundantly clear that the venoms produced among the colubrid rear-fanged snakes are
homologous with the much better characterized venoms of the front-fanged snakes. As trophic
adaptations that facilitate feeding, venoms vary in composition with several important factors,
including phylogeny, and so it is to be expected that among the diverse colubrid lineages, novel
compounds, and new functional variants of better-known venom proteins, will be encountered.
Much progress toward understanding rear-fanged snake venom composition has been made in the
last decade, but, as indicated above, we have barely begun to explore the diversity of advanced snakes
that comprise the colubrids. Transcriptomic and genomic approaches will greatly facilitate this work,
but it must be remembered that functional assays should also accompany analysis of any venom,
because the common recurring motif in venom biochemistry is to make the most of a stable molecular
scaffold, perhaps best exemplified by the varied pharmacologies of the three-finger toxin superfamily.
These small, structurally conservative peptides have very similar crystal structures but affect systems
as diverse as neurotransmission, the blood clot cascade, ion channel function, and salamander limb
regeneration and courtship. As Dr. Jay Fox once said, in venoms “we find only what we are looking
Toxins 2016, 8, 230 17 of 24

for”, and, to find truly novel toxins that will likely be present in some colubrid venoms, we will have
to look beyond the “normal” families of venom proteins.

4. Materials and Methods

4.1. Original Transcriptomic Data

4.1.1. Animals
Three specimens of Erythrolamprus miliaris (one male and two females) five specimens of
Oxyrhopus guibei (two males and three females) and two specimens of Xenodon merremi (both female)
were provided by the Laboratory of Herpetology at the Instituto Butantan. These animals were
collected from the wild by the local population, delivered at Instituto Butantan and kept in captivity for
a short time (up to one month); all snakes were provided water ad lib but not fed. Manual extraction of
the venom was performed 4 days prior to euthanizing the animals and dissecting out both Duvernoy’s
venom glands, which were frozen in liquid nitrogen. All animal procedures were authorized by
the Ethical Committee for Animal Research of Butantan Institute (protocols 164/2004 and 935/12,
approved on 11 May 2004 and 1 June 2012, respectively), according to principles adopted by the
Brazilian College of Animal Experimentation.

4.1.2. RNA-Seq
Erythrolamprus miliaris and Oxyrhopus guibei transcriptomes were investigated using RNA-Seq,
in a 454 pyrosequencing platform. Pairs of glands from each specimen were ground into a powder
in liquid nitrogen and homogenized using a Polytron Tissue Homogenizer (Kinematica, Luzern,
Switzerland). Total RNA was extracted with TRIZOL Reagent (Life Technologies, Thermo Fisher
Scientific, Carlsbad, CA, USA) and mRNA was prepared using the Dynabeads mRNA DIRECT kit
(Life Technologies, Thermo Fisher Scientific, Carlsbad, CA, USA). mRNA was quantified by the
Quant-iTTM RiboGreen RNA reagent and kit (Life Technologies, Thermo Fisher Scientific, Carlsbad,
CA, USA). To obtain 500 ng of mRNA needed to prepare cDNA libraries for pyrosequencing with cDNA
Synthesis System kit (Roche Diagnostics, Basel, Switzerland), we pooled mRNAs from individual
specimens of each species. Emulsion PCR amplification and library sequencing were performed
individually for each species, using a GS Junior 454 Sequencing System (Roche Diagnostics, Basel,
Switzerland) according to the manufacturer’s protocols. The raw sequences were deposited in GenBank
SRA with the accession numbers SRR3141951-SRR3141952 (Erythrolamprus miliaris) and SRR3141953
(Oxyrhopus guibei).
The raw reads from each species were assembled with Newbler 2.7 (Roche Diagnostics, Basel,
Switzerland), which first removes adaptors and contaminating ribosomal RNA sequences. The assembly
parameters were set to: (i) a minimum overlap length of 50% of the read; and (ii) a minimum overlap
identity of 98%, with all other parameters set as default. The resulting unigenes were deposited
in the GenBank TSA repository with the accession numbers GEFK00000000.1 linked to Bioproject
PRJNA310611 (Erythrolamprus miliaris) and GEFL00000000.1 linked to Bioproject PRJNA310661
(Oxyrhopus guibei). Unigene sequences were automatically annotated using Blast2Go [101] by
performing a Blast search against the UniProt database with the algorithm BlastX to identify similar
sequences. Toxin categories were manually assigned by comparing the unigenes to a compiled list of
known snake toxins. Final manual curation of relevant unigene sequences was undertaken to improve
the quality and the extension of the automatically assembled unigenes. The levels of expression of
individual unigenes were calculated using the RNA-Seq function of CLC Genomics Workbench v8
(Qiagen, Hilden, Germany, 2015)) by mapping cleaned reads (without known contaminants and
rRNAs) back to the unigenes and normalizing the count number by the length of the unigene using
RPKM (reads per kilobase per million of mapped reads) formula [102].
Toxins 2016, 8, 230 18 of 24

4.1.3. Expressed Sequence Tags (ESTs) Generation

Xenodon merremi transcriptome was investigated by means of EST generation, prior to the
common use of NGS (next generation sequencing). The pairs of glands from Xenodon merremi
specimens were ground into a powder in liquid nitrogen and homogenized using a Polytron Tissue
Homogenizer (Kinematica, Luzern, Switzerland). Total RNA was extracted with TRIZOL reagent
(Life Technologies, Thermo Fisher Scientific, Carlsbad, CA, USA) and mRNA was prepared using a
column of oligo-dT cellulose (GE). To obtain 5 µg of mRNA needed to prepare cDNA libraries using
the Superscript Plasmid System for cDNA Synthesis and Cloning (Life Technologies, Thermo Fisher
Scientific, Carlsbad, CA, USA), we pooled mRNAs from the two specimens. The cDNA was ligated
with the adaptors included in the kit, size selected into two ranges (250–600 bp and over 600 bp),
directionally cloned into pSPORT-1 plasmids and transformed in E. coli DH5α electrocompetent
cells. Plasmid DNA was isolated using alkaline lysis and sequenced on an ABI 3100 sequencer using
the BigDye 3.1 kit (Applied Biosystems, Foster City, CA, USA) with a standard 51 primer (M13R).
The electropherogram files were analyzed in a semi-automatic way and then assembled in clusters
of contiguous sequences using the CAP3 program [103] set for 98% or more of base identity in a
high-quality region. The resulting unigenes were deposited in GenBank TSA repository with the
accession number GETV00000000 linked to Bioproject PRJNA310192. The relative representation of
each cluster was given by the number of ESTs used in its assembly, as described elsewhere [104].

4.2. Public Database Sequence Retrieval

Prototypical sequences of the different kinds of proteins known in the venoms of Viperidae,
Elapidae, Atractaspididae and Colubridae snakes were compiled from GenBank and from our
archives and used as in silico probes to a more extensive search for related protein sequences from
Colubroidea snakes. Searches were performed using a stand-alone Blast tool of CLC Genomics
Workbench v8 against a downloaded version of the GenBank nr database (December 2015),
which includes non-redundant protein sequences from GenBank database and protein sequences
from TSA (transcriptome shotgun assembly) repository. An initial e-value cutoff of lower than 10´5
was considered but the alignment of each sequence identified was individually evaluated to decide for
the retrieving of a given protein. Whenever possible, the expression level of the transcript coding for
each protein was examined from the bibliographical reference associated with the sequence to assign
the “T” or “t” symbols on the summarizing tables, corresponding, respectively, to “high” (meaning
highly expressed or higher expressed than in other tissues) or “low” (meaning lowly expressed or lower
expressed than in other tissues) transcriptional level in the venom glands. Evidence for proteomic
identification of proteins was also obtained from the literature.

4.3. Sequence Comparisons and Evolutionary Analyses

Protein sequences were incorporated into gene family sequence alignments containing toxin
and non-toxin protein homologs and paralogs and representative outgroup sequences. Alignments
were performed using MUSCLE or CLUSTALW tools of CLC Genomics Workbench v8 (Qiagen) and
checked manually. Phylogenetic trees were generated by the Maximum Likelihood method, with WAG
substitution model and bootstrapping 1000 replicates, using CLC Genomics Workbench.

Supplementary Materials: The following are available online at www.mdpi.com/2072-6651/8/8/230/s1,

Table S1: Assembled contigs from the species investigated and their expression values.
Acknowledgments: This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São
Paulo (FAPESP, grant numbers 12/00177-5, 2013/07467-1) and Conselho Nacional de Desenvolvimento Científico
e Tecnológico (CNPq) to Inácio L.M. Junqueira-de-Azevedo and Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior (fellowship 387422) to Pollyanna Fernandes Campos.
Author Contributions: I.L.M.J.-d.-A. conceived the research, analyzed sequences and wrote the paper; P.F.C. and
A.T.C.C. performed the transcriptomic experiments and analyzed sequences; and S.P.M. discussed the results and
wrote the paper.
Toxins 2016, 8, 230 19 of 24

Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

3FTx three finger toxin

5NUCLEO 51 nucleotidase
AChE acetylcholinesterase
AVIT AVIT protein
bPLA2i beta type phospholipase A2 inhibitor
CDS coding DNA sequence
CNP C-type natriuretic peptide
CRISP cysteine rich secretory protein
CTL C-type lectin
DEFEN defensin (crotamine-like)
CVF cobra venom factor
CYST cystatins
DEF defensin
DPP dipeptidyl peptidase
EGFr EGF repeats protein
ESTs expressed sequence tags
FactV venom coagulation factor V
FactX venom coagulation factor X
gPLA2 i gamma type phospholipase A2 inhibitor
HYAL hyaluronidase
KUN-1 kunitz type protein (type 1)
KUN-2 kunitz type protein (type 2)
KU-WA-FU ku-wap-fusin protein
LAAO L -amino acid oxidase
Lactha lactadherin-like protein
LIPA snake venom acid lipase
LIPO lipocalin
NGF nerve growth factor
OHA ohanin (vesprin) protein
PDE phosphodiesterase
PLA2 (IA) phospholipase A2 (type IA)
PLA2 (IIE) phospholipase A2 (type IIE)
PLB phospholipase B
RPKM reads per kilobase per million of mapped reads
svMMP snake venom matrix metalloproteinase
SVMP snake venom metalloproteinase
SVSP snake venom serine proteinase
TSA transcriptome shotgun assembly
Vefico veficolin (ficolin-like)
VEGF-A vascular endothelial growth factor (type A)
WAP waprin-like proteins

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